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EP0486572B1 - Rezeptor für granulozyten-macrophagen-koloniestimulierungsfaktor und seine derivate - Google Patents

Rezeptor für granulozyten-macrophagen-koloniestimulierungsfaktor und seine derivate Download PDF

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Publication number
EP0486572B1
EP0486572B1 EP90912329A EP90912329A EP0486572B1 EP 0486572 B1 EP0486572 B1 EP 0486572B1 EP 90912329 A EP90912329 A EP 90912329A EP 90912329 A EP90912329 A EP 90912329A EP 0486572 B1 EP0486572 B1 EP 0486572B1
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Prior art keywords
csf
receptor
cells
binding
hgm
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EP0486572A4 (en
EP0486572A1 (de
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Nicos Anthony Nicola
Nicholas Martin Gough
David Paul Gearing
Donald Metcalf
Julie Ann King
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CSL Innovation Pty Ltd
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Amrad Corp Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present invention relates generally to human recombinant and synthetic granulocyte-macrophage colony stimulating factor (GM-CSF) receptor and to biochemical and/or biological equivalents, homologues or derivatives thereof. These molecules are useful inter alia in the preparation of therapeutics and diagnostics and in the generation of agonists and antagonists with respect to the binding of GM-CSF to its receptor.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Granulocyte-macrophage colony-stimulating factor is a glycoprotein growth and differentiation factor which regulates the proliferation, differentiation and functional activity of cells of the neutrophil, eosinophil and monocyte/macrophage series (reviewed in Metcalf, 1984, and Gough and Nicola, 1989).
  • GM-CSF has been found to elevate circulating levels of monocytes, neutrophils and eosinophils; to enhance the functional capacities of the circulating cells; and to enhance the rate of haemopoietic recovery following chemotherapy and/or bone marrow transplantation (Gough and Nicola, 1989; Morstyn et al, 1989).
  • GM-CSF receptors are present in low numbers (a few hundred per cell) on cells within the monocyte, neutrophil and eosinophil lineages (Nicola, 1987; DiPersio et al, 1988).
  • functional GM-CSF receptors have also been detected on non-haemopoietic cells, including endothelial cells (Bussolino et al, 1989), small-cell lung carcinoma cell lines and SV40-transformed simian COS cells (Cocita Baldwin et al, 1989).
  • Multi-CSF interkeukin-3
  • GM-CSF receptors can down-modulate GM-CSF receptors on some types of hemopoietic cells, but it is not clear whether this is mediated by different receptor subclasses or by receptor-receptor interactions (Gearing et al, 1989; Elliot et al , 1989; Lopez et al , 1989).
  • GM-CSF was originally defined by its ability to stimulate the proliferation of granulocyte/macrophage progenitor cells, but more recently it has become apparent that it can also stimulate the proliferation of progenitor cells of other haemopoietic lineages (Metcalf et al, 1980) and cells of non-haemopoietic origin.
  • the latter include human bone marrow fibroblasts, osteogenic sarcoma cell lines and a breast carcinoma cell line (Dedhar et al, 1988), human small cell carcinoma cell lines (Cocita Baldwin et al, 1989), human endothelial cells (Bussolino et al , 1989) and human placental cells (Wegmann et al , 1989).
  • GM-CSF stimulates human endothelial cell migration (Bussolino et al, 1989), the proliferation and function of human osteoblast-like cells in vitro (Evans et al , 1989) and improves the growth of murine placental cells in vivo (Wegmann et al, 1989).
  • GM-CSF receptors detected on these cells are functional, despite the fact that only high affinity receptors were detected on the endothelial cells, whereas only low affinity receptors were detected on the fibroblasts and placental membranes.
  • GM-CSF receptor Although some of the properties of GM-CSF receptor have been deduced, the receptor has not hitherto been isolated or purified.
  • a purified nucleic acid selected from the group consisting of single stranded DNA, double stranded DNA, cDNA and RNA which each comprises a nucleotide sequence which encodes, or is complementary to a nucleotide sequence which encodes, human GM-CSF receptor or a derivative thereof capable of binding human GM-CSF, said GM-CSF receptor being defined as a glycosylated or unglycosylated proteinaceous molecule comprising, in its entirety, an extracellular domain, a transmembrane domain, and an intracytoplasmic tail and being capable of specifically binding radioactively labelled GM-CSF or derivatives thereof, said binding being competed for by unlabelled GM-CSF.
  • the expression of this human GM-CSF gene provides a quantity of recombinant receptor heretofore unavailable, thereby permitting inter alia the development of receptor therapeutics, diagnostics, agonists and antagonists, and the like.
  • the first aspect of the present invention relates to recombinant or synthetic human GM-CSF receptor or a derivative thereof capable of binding human GM-CSF, essentially free of other proteins, said GM-CSF receptor being defined as a glycosylated or unglycosylated proteinaceous molecule comprising, in its entirety, an extracellular domain, a transmembrane domain, and an intracytoplasmic tail and being capable of specifically binding radioactively labelled GM-CSF or derivatives thereof, said binding being competed for by unlabelled GM-CSF.
  • Derivatives included in this aspect of the invention are those including that portion of the receptor comprising its soluble (non-membrane associated) region that has the capacity to bind GM-CSF.
  • a second aspect of the present invention relates to antibodies that recognise human recombinant or synthetic GM-CSF receptor (and its derivatives) as hereinbefore defined, and which are useful in the detection and/or purification of these molecules.
  • the binding of GM-CSF to its cell-bound receptor may be modulated with agonists or antagonists of GM-CSF.
  • the receptors of the present invention may be used in a method of treatment of GM-CSF related diseases in a mammal, and in particular a human, by the administration to said mammal of an effective amount of recombinant or synthetic GM-CSF receptor or its derivative.
  • One such method involves modulating the proliferation, differentiation or functional activation of GM-CSF-stimulation-sensitive cells in the mammal, which method comprises the administration to said mammal of an effective amount of recombinant or synthetic GM-CSF receptor of its derivative for a time and under conditions sufficient to reduce the amount of non-bound GM-CSF.
  • an effective amount can readily be determined by routine experiment, as can the most effective and/or convenient route of administration, such as intravenous, intramuscular, subcutaneous, or oral. Slow release formulations may be advantageous for specific purposes.
  • the mammal and the origin of the GM-CSF receptor are homologous, and even more preferably the homologous system is human.
  • a third aspect of the present invention provides a method of generating information for use in the diagnosis of a GM-CSF related disease in a mammal, comprising the step of contacting a tissue or biological fluid or said mammal, in vitro, with a nucleic acid or recombinant DNA molecule, a recombinant cell containing such a molecule, a recombinant or synthetic GM-CSF receptor or an antibody or antigen-binding fragment thereof as hereinbefore defined, and detecting the thus-formed product.
  • the invention also extends to kits for such diagnostic purposes. For example, diagnostic kits for assays utilizing radioimmunoassays, fluorescent immunoassay, or ELISA are specifically contemplated.
  • a fourth aspect of the present invention relates to the use of recombinant or synthetic human GM-CSF receptor or an antibody or antigen binding fragment thereof as hereinbefore defined in the manufacture of a medicament for the treatment of GM-CSF related diseases.
  • a fifth aspect of the invention relates to a GM-CSF-dependent haemopoietic cell line into which a low-affinity receptor for a heterologous GM-CSF has been cloned.
  • a sixth aspect of the invention relates to a method of screening a cDNA library for a cDNA fragment encoding GM-CSF receptor, comprising the steps of: constructing a cDNA library, preparing cDNA fragments therefrom, transfecting said fragments into mammalian hosts cells, incubating said transfected cells with labelled GM-CSF, identifying populations of transfected cells binding said labelled GM-CSF, preparing clones of host cells transformed with cDNA fragments able to cause mammalian host cells to bind labelled GM-CSF, and isolating said clones.
  • Figure 1 shows saturation binding isotherms and Scatchard analysis of 125 I-hGM-CSF binding to
  • FIG. 2 shows the binding specificity of hGM-CSF and h-IL-3 receptors on human placental membranes.
  • Membranes 40 p1 were incubated in duplicate with 125 I-hGM-cSF (200,000 cpm in 110 ⁇ l HRF) with or without unlabelled hGM-CSF (500 ng) or h-IL-3 (100 ng) for 1 hour at 20°C.
  • 40 ⁇ l membranes were incubated with 125 I-h-IL-3 (220,000 cpm in 110 ⁇ l HRF) with or without the same amounts of unlabelled hGM-CSF or IL-3 as above.
  • Total binding to each membrane preparatlon (mean ⁇ range) is shown.
  • Figure 3 shows the detection and specificity of hGM-CSF receptors transfected to COS-7 cells.
  • FIG. 4 shows saturation and competition binding analysis of the placental hGM-CSF receptor transfected to COS-7 cells.
  • COS-7 cells (33,000 per point) transfected 48 hours earlier with the pure clone (pGMR138) were incubated with increasing concentrations of 125 I-hGM-CSF (200,000 cpm) (Panel A) or a constant amount of 125 I-hGM-CSF and increasing concentrations of unlabelled hGM-CSF or h-IL-3 (Panel B) in a constant volume of 85 ⁇ l HRF/20 mM EDTA/100 ⁇ g/ml chondroitin sulphate at 20°C for 1.5 hours.
  • Panel A total binding, non-specific binding and specific binding are shown with Scatchard transformation of the specific binding data shown underneath.
  • Panel B total binding is shown in the upper panel, with Scatchard transformation of the specific binding data shown underneath.
  • Figure 5 shows analysis of chemical cross-linking of the hGM-CSF receptor on DMSO-treated HL60 cells and on transfected COS-7 cells, using SDS-polyacrylamide gel electrophoresis.
  • tracks A-D 5x10 6 HL60 cells treated for 9 days with DMSO were used per point while in tracks E-J, 7x10 4 transfected COS-7 cells were used per point.
  • binding was for 3 hours at 4°C with 125 I-hGM-CSF at 2 nM.
  • Tracks C and D were with or without 10 min dissociation in 1 ml PBS to remove low-affinity binding, and A and B are as for C and D except that 20 nM unlabelled hGM-CSF was included during the binding reaction.
  • Figure 6(A) represents a restriction endonuclease cleavage map of the insert cDNAs of pGMR138 and pGMR29. Boxes represent open reading frames. The hatched and filled regions represent the signal sequence and transmembrane region of the GM-CSF-R coding region, respectively. The stippled box represents the upstream open reading frame.
  • Figure 6(B) shows the combined nucleotide sequence and deduced amino acid sequence of the insert cDNAs of pGMR138 and pGMR29. Numbers at the right margin indicate positions of nucleotides and numbers above the sequence refer to the amino acid sequence.
  • the hatch marks indicate potential N-glycosylation sites (Asn-X-Ser/Thr).
  • the overlined regions indicate the presumed signal peptide and transmembrane region, respectively.
  • the sets of six asterisks identify possible poly(A) addition signals.
  • the poly(A) tail in clone 138 was 61 nucleotides long.
  • Figure 8 is a photograph showing the detection of the human GM-CSF receptor transcript.
  • Figure 9 depicts the alignment of the sequence of the hGM-CSF-receptor with other growth factor receptors. Amino acids are identified using the single letter abbreviations standard in the art.
  • Figure 10 shows viral integration and transcripts in hGM-R-FD cells.
  • Figure 11 illustrates the responsiveness of cells from cloned hGMR-FD cell lines to proliferative stimulation by recombinant m- or hGM-CSF.
  • Lines maintained in human GM-CSF exhibit a similar content of clonogenic cells with both stimuli (eg. clone 54), whereas in lines maintained in m+hGM-CSF (eg. clone 21) murine-responsive clonogenic cells are more frequent than human-responsive cells.
  • clonogenic cells are less responsive to human than to murine GM-CSF.
  • Figure 12 shows saturation binding analysis and Scatchard transformation of 125 -hGM-CSF binding to hGM-R-FD cell clones.
  • Figure 13 illustrates the internalization of 125 I-hGM-CSF bound to hGM-R-FD clone 33 cells (upper curve) or hGM-R-FD clone 53 cells (lower curve) at 37°C.
  • the former cells had been maintained in a mixture of murine and human GM-CSF while the latter had been maintained in hGM-CSF only.
  • the curves show the variation of cell surface-associated and internalized radioactivity with time, after addition of 125 I-hGM-CSF, determined as described (Nicola et al, 1988); the lines through the experimental points (means of duplicate tubes) were fitted by computer as described by Nicola et al (1988).
  • clone 33 1.9x10 6 cells were used per point and the 125 I-hGM-CSF concentration was 13nM, and for clone 53, 1.8x10 6 cells were used per point and the 125 I-hGM-CSF concentration was 13nM.
  • GM-CSF receptor is meant a glycosylated or unglycosylated proteinaceous (i.e. amino acid containing) molecule comprising, in its entirety, an extracellular domain, a transmembrane domain and an intracytoplasmic tail.
  • the receptor molecule is capable of specifically binding radioactively labelled GM-CSF or derivatives thereof, said binding being defined inter alia as being competed for by unlabelled GM-CSF.
  • recombinant GM-CSF receptor is meant a glycosylated or unglycosylated polypeptide molecule, with or without other associated molecules (eg lipids), produced by recombinant means such as by the ligation of a cDNA molecule encoding the receptor or its derivative into an appropriate expression vector in the correct reading frame relative to a promoter, introducing the resultant recombinant expression vector into a suitable host and growing said host under conditions appropriate for the expression and, if necessary, transportation of the recombinant receptor or its derivative from said host, and then purifying the recombinant receptor or derivative.
  • recombinant means such as by the ligation of a cDNA molecule encoding the receptor or its derivative into an appropriate expression vector in the correct reading frame relative to a promoter, introducing the resultant recombinant expression vector into a suitable host and growing said host under conditions appropriate for the expression and, if necessary, transportation of the recombinant receptor or its derivative from said host,
  • GM-CSF-stimulation-sensitive cell a cell carrying a receptor to which GM-CSF can bind thereby causing the stimulation of proliferation or functional activation of that cell, as hereinbefore described.
  • bind in relation to GM-CSF and its receptor is used in its broadest sense, and means any association between GM-CSF and its receptor, and particularly in relation to cell-bound receptor, sufficient to induce the stimulation of proliferation or functional activation of the cell on which the receptor is located.
  • Non-bound GM-CSF generally means circulating GM-CSF.
  • substantially amino acid homology molecules having a sequence homology of approximately 75% or more, preferably greater than or equal to 85% and even more preferably greater than or equal to 90-95%.
  • Cancer is used in its broadest sense, and includes cancers, tumours and leukaemias en masse or as individual cells. "Aberrations" of bound GM-CSF receptor or its nucleotide sequence is used to mean any alteration in amino acid and/or nucleotide sequence detectable by analysis of the respective homologues as determined, for example, by hybridization studies. Accordingly, a cancer cell may contain an altered GM-CSF receptor detectable as being so altered by the use of the GM-CSF receptor or its encoding nucleotide sequence contemplated by the present invention.
  • the subject cDNA into a prokaryotic expression vector and express same in bacteria, such as Escherichia coli, Bacillus sp. or Pseudomonas species.
  • bacteria such as Escherichia coli, Bacillus sp. or Pseudomonas species.
  • the cDNA can be expressed in eukaryotic cells such as yeasts, fungi, insect cell lines, mammalian cell lines other than COS cells, or plant cells.
  • the cDNA can also be transferred into germ line or somatic cells to form transgenic animals.
  • the present invention extends to single or double stranded DNA, cDNA or mRNA wherein at least one nucleotide strand thereof encodes, or is complementary to, a nucleotide strand which encodes human GM-CSF receptor or its derivatives, and to any vector, expression or otherwise, containing same, including viral vectors.
  • the invention provides a recombinant DNA molecule comprising a gene encoding the human low-affinity GM-CSF receptor, or a homologue thereof which encodes a polypeptide having a least 75% sequence identity with said receptor and which retains at least one-tenth of the relative binding affinity of the native human low-affinity GM-CSF receptor for human GM-CSF, said gene being operably linked to a promoter. More preferab said polypeptide comprises an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the sequence presented in Figure 6B refers specifically to the low-affinity GM-CSF receptor
  • the evidence presented herein indicates that the low affinity GM-CSF receptor itself forms a component of the high-affinity GM-CSF receptor, which may be a multimer of the low-affinity GM-CSF receptor.
  • the high-affinity GM-CSF receptor is therefore specifically included in the scope of the present invention.
  • the GM-CSF receptor molecule having the amino acid sequence shown in Figure 6B.
  • This molecule is a 400 amino acid polypeptide with a molecular weight of approximately 45,000, having a single hydrophobic transmembrane domain, a glycosylated extracellular domain and a short (54 amino acid) intracytoplasmic tail.
  • the ligand-binding domain (23-319) contains eleven cysteine residues.
  • the subject receptor does not contain a tyrosine kinase domain, and does not show homology with members of the immunoglobulin gene superfamily, but does share sequence homology with receptors of other haemopoietic growth factors such as human IL-6, erythropoietin and IL-2 ( ⁇ -chain).
  • the present invention includes within its scope a synthetic GM-CSF receptor molecule prepared by the chemical addition of amino acids by established techniques and in a sequence as established herein.
  • the invention further includes recombinant or synthetic derivatives of GM-CSF receptor carrying single or multiple amino acid substitutions, deletions and/or additions to any or all of the aforementioned regions or domains of the receptor molecules.
  • Such derivatives may be functional (i.e. biological) equivalents with respect to their ability to bind to GM-CSF or its derivatives, and/or may exhibit substantial amino acid homology to the aforementioned GM-CSF receptor amino acid sequence.
  • One preferred derivative of GM-CSF receptor comprises all or part of the extracellular domain (soluble portion).
  • Derivatives also include any or all of the receptor molecule in glycosylated or unglycosylated form.
  • the recombinant receptor may or may not be glycosylated. Both glycosylated and unglycosylated forms of the recombinant or synthetic GM-CSF receptor or its derivatives are within the scope of the present invention. Functionally active derivatives or equivalents of the GM-CSF receptor can readily be identified using the methods described herein.
  • the invention is also directed inter alia to the cDNA encoding a GM-CSF receptor as hereinbefore defined.
  • the cDNA comprises a nucleotide sequence shown in Figure 6B.
  • the scope of the present invention includes cDNA derivatives carrying single or multiple nucleotide substitutions, deletions and/or additions relative to the aforementioned cDNA sequence. Such derivatives may encode the entire GM-CSF receptor molecule or derivatives thereof, such as a GM-CSF receptor carrying single or multiple amino acid substitutions, deletions and/or additions.
  • the present invention also encompasses a cDNA carrying a nucleotide sequence substantially homologous to the subject nucleotide sequence, i.e.
  • the present invention further includes molecules such as polypeptides fused to the GM-CSF receptor or its derivatives, or nucleotide sequences contiguous to GM-CSF receptor-encoding sequences.
  • molecules such as polypeptides fused to the GM-CSF receptor or its derivatives, or nucleotide sequences contiguous to GM-CSF receptor-encoding sequences.
  • a fusion protein comprising GM-CSF receptor or its derivative and an amino acid sequence from another polypeptide or protein
  • examples of the latter especially prokaryotic systems, being enzymes such as ⁇ -galactosidase, phosphatase, urease and the like.
  • Most fusion proteins are formed by the expression of a recombinant gene in which two coding sequences have been joined together such that their reading frames are in phase.
  • polypeptides can be linked in vitro by chemical means. All such fusion or hybrid derivatives of GM-CSF receptor or the respective encoding nucleotide sequence
  • the present invention therefore, provides recombinant and synthetic GM-CSF receptor and derivatives thereof in sufficient quantity for the development of, for example, receptor therapeutics and diagnostics.
  • another aspect of the invention contemplates a method for modulating the proliferation or functional activation of GM-CSF-stimulation-sensitive cells in a mammal which method comprises the administration to said mammal of an effective amount of GM-CSF receptor or its derivative for a time and under conditions sufficient to reduce the amount of non-bound GM-CSF.
  • the subject method is predicated on the binding of GM-CSF circulating in the body to soluble receptor before the lymphokine can bind to the cell-bound receptor, thereby reducing the amount of GM-CSF available for binding to GM-CSF-stimulation-sensitive cells.
  • the invention extends to the use of the entire recombinant or synthetic GM-CSF receptor or its derivative in the modulation of proliferation or functional activation of GM-CSF-stimulation-sensitive cells, but preferably the soluble or extracellular domain is used.
  • This method is particularly useful in treating disease states such as myeloid leukaemias where the leukaemic cells express GM-CSF receptors (Young and Griffin, 1986). Many types of myeloid leukaemia cells, for example, cannot undergo proliferation in vitro unless exogenous colony stimulating factors are available (Metcalf, 1984). Accordingly, recombinant or synthetic GM-CSF receptor or its derivatives administered in vivo will bind to circulating GM-CSF and thus compete for binding of the latter to cell bound GM-CSF receptor. The subject method will also assist as therapeutic agents in abrogating the toxic effects of over-production of GM-CSF noted in animal model systems (Lang et al, 1987; Johnson et al, 1989).
  • the present invention also extends to antibodies, and particularly monoclonal antibodies, to recombinant or synthetic GM-CSF receptor or its derivatives. Such antibodies are particularly useful in purifying GM-CSF receptors and for quantitating the same.
  • the subject invention also extends to antibodies (monoclonal or polyclonal) against said first antibodies for the purposes of assaying plasma, serum or body fluids, cell surfaces or cell extracts for the presence of GM-CSF receptors.
  • One or other of the aforementioned antibodies may be labelled with a reporter molecule for use, for example, in sandwich assays.
  • Methods for production and screening of polyclonal and monoclonal antibodies optionally including the use of adjuvants, methods for labelling such antibodies with radioactive, fluorescent, or chemical labels, and methods of immunoassay, such as radioimmunoassay, fluorescent immunoassay, and enzyme-linked immunoassay (ELISA), and coupling of antibodies to solid supports to form immunoadsorbents, are routine in the art.
  • adjuvants methods for labelling such antibodies with radioactive, fluorescent, or chemical labels
  • immunoassay such as radioimmunoassay, fluorescent immunoassay, and enzyme-linked immunoassay (ELISA)
  • ELISA enzyme-linked immunoassay
  • the recombinant or synthetic GM-CSF receptors can be used in developing agonists and antagonists to augment or reduce binding of GM-CSF to its cell-bound receptors.
  • a transfected cell line bearing recombinant GM-CSF receptors may be used to screen compounds for such activities.
  • the present invention extends to the use of such agonists or antagonists in the treatment of disease states caused by excessive or insufficient proliferation or functional activation of GM-CSF-stimulation-sensitive cells.
  • Possible agonists or antagonists include naturally occurring or synthetic fragments of GM-CSF, as well as other naturally occurring or synthetic chemical compounds, which may be screened by the aforesaid method, using binding of labelled GM-CSF to the cells as a marker.
  • the present invention also extends to the use of GM-CSF or its derivatives in the manufacture of a medicament for the treatment of GM-CSF-related diseases.
  • diseases include cancers, tumors and leukaemias caused by or associated with GM-CSF-stimulation-sensitive cells.
  • Another aspect of the present invention extends to the use of recombinant or synthetic GM-CSF receptor or derivatives thereof, and particularly to nucleotide sequences encoding same, in the diagnosis of cancers composed of, or associated with, GM-CSF-stimulation-sensitive cells by detecting cell-bound GM-CSF receptors or aberrations thereof, or nucleotide sequences encoding same, in said cancer cells.
  • the functional, low-affinity hGM-CSF receptor has been cloned from placenta, and it has been shown that it recognizes only GM-CSF and not interleukin-3.
  • This receptor when transfected into COS cells, shows nearly identical low affinity to, and the same specificity as the receptor on placental membranes.
  • Trp236 is conserved, and in three of the receptors is adjacent to an Arg residue.
  • a further homology between all four receptor sequences is found just N-terminal of the transmembrane domain ( Figure 9).
  • the consensus sequence starting at position 294 of the GM-CSF receptor (the "WS-WS" Dox) is found in all four receptors. The position of this sequence is close to the transmembrane domain in three of the receptors (for GM-CSF, erythropoietin and IL-2 ( ⁇ -chain)), but is further away in the IL-6 receptor.
  • the rPRL receptor also has a transmembrane cysteine residue (Boutin et al, 1988), but the transmembrane sequence is not homologous to the hGM-CSF receptor.
  • the "WS-WS” box is not found in a close relative of the prolactin receptor, the growth hormone receptor (Leung et al, 1988), which in other regions shares 75-100% sequence similarity with the prolactin receptor (Boutin et al, 1988).
  • the hGM-CSF receptor therefore appears to be a member of a new subset of growth and differentiation factor receptors, defined by the set of five receptors described above (i.e. hGM-CSF, hIL-6, murine EPO, hIL-2 ( ⁇ -chain) and rPRL receptors).
  • the monolayers were washed twice in medium, fixed in 2.5% (w/v) glutaraldehyde/PBS and dipped in 1% (w/v) gelatine as described (Nicola and Metcalf, 1985).
  • the slides were dipped in Kodak NTB2 photographic emulsion at 42°C and exposed in the dark for 48 hours at 4°C in light-proof boxes containing dessicant.
  • Slides were developed for 3 min in Kodak D19 developer (40g/500ml water), and rinsed in water and fixed for 3 min in Agfa G433C fixer before staining in 10% filtered Giemsa stain in water. Slides were screened at 10-20x magnification and two positive pools (#29 and #138) were selected.
  • Corresponding glycerol stocks of E.coli transformants were partitioned into smaller pools until single cDNA clones able to cause COS-7 cells to bind 125 I-GM-CSF were obtained.
  • Cytoplasmic polyadenylated RNA (approximately 1.5 ⁇ g), prepared essentially as described by Gough (1988), was fractionated on 1% (w/v) agarose gels containing 20mM morpholinopropane sulphonic acid, 5mM sodium acetate, lmM EDTA (pH 7.0), plus 6% (v/v) formaldehyde, and transferred to nitrocellulose.
  • RNA Prior to hybridization, filters containing RNA were soaked in 2xSSC containing 0.2% (w/v) Ficoll, 0.2% (w/v) polyvinylpyrollidone, 0.2% (w/v) bovine serum albumin, 2mM sodium pyrophosphate, 1mM ATP, 30 50 ⁇ g/ml denatured salmon sperm DNA and 50 ⁇ g/ml E. coli tRNA at 67°C for several hours. Hybridization was performed in the same buffer plus 0.1% (w/v) SDS at 67°C. The hybridization probe was the gel-purified 1300bp Xho 1 -Eco RI.
  • RNA (approx 1 ⁇ g) was subjected to first strand cDNA synthesis in a 20 ⁇ l reaction containing 50mM Tris-Cl (pH 8.3 at 42°C), 20mM KCl, 10mM MgCl 2 , 5mM dithiothreitol, lmM of each dNTP, 20 ⁇ g/ml oligo-dT 15 and 20 units AMV reverse transcriptase (Boehringer Mannheim) for 40 minutes at 42°C. After completion of first-strand synthesis, the reaction was diluted to 100 ⁇ l with distilled water and 5 pl used for each PCR (polymerase chain reaction).
  • Polymerase chain reactions contained 200 ⁇ M of each dNTP, 1 ⁇ M of each specific primer, buffer as supplied in the GeneAmp kit (Cetus Corp., USA) and 1.25 units Taq polymerase in a volume of 50 ⁇ l.
  • the primers used for PCR were 5'-CTTCTCTCTAGACCAGCA (position 131-147) and 5'-ACATGGGTTCCTGAGTC (position 676-660) defining a 530bp fragment.
  • the PCR reaction conditions were: 2 min at 94°C; 2 min at 65°C; 3 min at 72°C for 25 cycles in a Perkin-Elmer-Cetus DNA thermal cycler.
  • a portion of the PCR reaction was electrophoresed through a 1.2% (w/v) agarose gel and transferred to nitrocellulose. Filters were prehybridized, hybridized and washed as described above.
  • the hybridization probe was the gel-purified 1.9kbp cDNA insert of pGMR138, radiolabelled to a specific activity of approximately 10 9 cpm/ ⁇ g by random priming and included in the hybridization at approximately 2x10 7 cpm/ml.
  • the reaction mixture was passed through a column of Sephadex G-25M (Pharmacia, Uppsala, Sweden) to separate macromolecular radioactivity from free iodine (Hilton et al, 1989).
  • 125 I-hGM-CSF was 100% bindable (Calvo et al, 1983) and displayed a specific radioactivity of 20,000-40,000 cpm/ng by the self-displacement analysis of Calvo et al(1983).
  • 125 I-mGM-CSF had specific radioactivity 120,000 cpm/ng and bindability 40-50%.
  • Unlabelled and labelled (specific radioactivity 40,000 cpm/ng) human IL-3 was purchased from Amersham (Buckinghamshire, England).
  • HL60 cells grown for 5 days in DME medium/10% FCS containing 1.25% (w/v) dimethyl sulphoxide were resuspended at 5x10 6 cells/50 ⁇ l in Hepes (10mM, pH 7.2) buffered RPMI medium (HR) containing 10% (v/v) foetal calf serum (HRF).
  • HR RPMI medium
  • HRF foetal calf serum
  • the detached and disaggregated cells were centrifuged at 700 g for 5min and resuspended in HRF with or without 20 mM EDTA and 100 ⁇ g/ml chondroitin sulphate.
  • Saturation binding isotherms or competition experiments were performed as for HL60 cells.
  • Human placental membranes were prepared from fresh term placentas essentially as described by Yeung et al (1987), with 6g of placenta yielding 4 ml of membrane suspension. For each binding point, 40 ⁇ l of membrane suspension was mixed with 40 ⁇ l HRF and increasing concentrations of 125 I-hGM-CSF with or without excess unlabelled hGM-CSF (0.0 ⁇ M).
  • Binding of 125 I-hGM-CSF to cells in solution was performed at 4°C as described above, and the cell pellets resuspended in 1 ml of ice-cold Na phosphate buffered (20mM, pH 7.2) saline (0.15M).
  • Disuccinimidyl suberate (Sigma, Missouri, USA) in anhydrous acetonitrile (10 ⁇ l) was immediately added to give a final concentration of 0-lmM, and the cells incubated for 15 minutes on ice before centrifuging the cell pellet at 13,000 g for 1 minute.
  • the cell pellet was treated with DNAase in the presence of protease inhibitors and prepared for sodium dodecyl sulphate polyacrylamide gel electrophoresis as described (Nicola and Peterson, 1986).
  • Specific binding was determined as the difference between binding in the absence or presence of excess unlabelled hGM-CSF. Specifically bound cpm were converted to molar concentrations using the specific radioactivity of 125 I-GM-CSF determined by self-displacement analysis. Curve-fitting of the binding data was performed using the LIGAND program of Munson and Rodbard (1980) before conversion to the Scatchard transformation. Two binding site fits were used only if the fit to the data was significantly improved (p ⁇ 0.05) over the one binding site fit.
  • ⁇ 2 packaging cells Mann et al , 1983 were electroporated with pJZen2(SVNeo)-hGM-R DNA as previously described (Johnson et al, 1989), and transfectants were selected 2 days later using 400pg/ml of antibiotic G418 (Geneticin, Sigma). G418-resistant 2 clones were selected for high surface expression of hGM-R by bindlng of 125 I-hGM-CSF. Retroviral titres of receptor-positive ⁇ 2 clones were tested by polybrene-mediated infection of NIH3T3 fibroblasts (Cepko et al, 1984). The clone selected for further work ( ⁇ 2-GMR) had a titre of 1.2x10 4 viral particles/ml.
  • Adherent ⁇ 2-GMR cells (3x10 6 per 75cm 2 flask) were irradiated (35 Gy), and co-cultured with 10 6 FDC-P1 cells (Dexter et al, 1980) in 20 ml Dulbecco's Modified Eagle's Medium (DMEM) wlth 10% foetal calf serum (FCS) and 10% pokeweed-mitogen stimulated spleen cell conditioned medium. Washed supernatant cells from 48 hour cocultures were cultured in agar-medium at a density of 300 cells/ml with either 10 3 U/ml of mGM-CSF, 6x10 3 U/ml of hGM-CSF, or a combination of both.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS foetal calf serum
  • pokeweed-mitogen stimulated spleen cell conditioned medium Washed supernatant cells from 48 hour cocultures were cultured in agar-medium
  • clones developing in mGM-CSF had achieved 50-100 cells in size. In contrast, fewer clones had developed in cultures stimulated by hGM-CSF; these were dispersed in morphology, and most contained only 10-30 cells.
  • Individual clones growing in cultures stimulated by hGM-CSF were removed using a micropipette, and cloned cell lines established and maintained in 1 ml cultures of DMEM with 20% FCS containing either 6x10 5 U/ml hGM-CSF (12 lines) or 6x10 3 U/ml hGM-CSF plus 10 3 U/ml mGM-CSF (36 lines). Subcloning of individual cell lines was performed by growing colonies in agar medium cultures of 200 cells/ml then removing individual colonies after 7 days of incubation and continuing culture of these colonies in suspension.
  • the stimuli used for colony formation were purified recombinant mGM-CSF (specific activity 3x10 8 U/mg protein) or purified recombinant hGM-CSF (specific activity 10 8 U/mg protein) produced as nonglycosylated derivatives in E.coli. These were included as 0.lml volumes during preparation of the agar cultures, and serial twofold dilutions were performed using 5% FCS in 0.95% saline. Mean cell numbers in 7-day colonies were determined by pooling 30-50 sequential colonies.
  • both high and low affinity GM-CSF receptors were detected on human bone marrow cells, primary human myeloid leukaemic cells and the human promyelocytic leukaemic cell line, HL-60 (Fig. 1).
  • Fig 1A shows the saturation binding isotherm at 4°C for 125 I-hGM-CSF binding to HL-60 cells that had been induced to differentiate for 5 days in 1.25% (w/v) dimethylsulphoxide (DMSO).
  • DMSO dimethylsulphoxide
  • the human placental GM-CSF receptor appeared to recognize only hGM-CSF and not h-IL-3 (Fig 2).
  • no hIL-3 receptors were detected on human placental membranes (Fig 2).
  • this procedure is extremely sensitive, since single clones could be detected from pools of 2x10 4 clones (c.f. 1 in 10 3 , D'Andrea et al, 1989; 1 in 350, Sims et al, 1988).
  • the procedure allows easy identification of artefacts due to non-specific binding, which would otherwise have made identification of a single GM-CSF-receptor positive cell on a slide containing approximately 10 6 negative COS cells impossible.
  • a human placental cDNA library of approximately 5x10 6 independent recombinants in a COS cell expression vector was sub-fractionated into 500 pools of approximately 2x10 4 clones each, and DNA from each pool was separately transfected into 1.5x10 6 COS cells by electroporation.
  • the cells were cultured for 48 hours on glass slides, incubated with a relatively high concentration of 125 I-hGM-CSF (approximately 2nM), fixed and then dipped in liquid photographic emulsion.
  • the slides were developed and individually examined microscopically; positive cells were identified by the presence of autoradiographic grains. Of the first 250 pools screened, two gave rise to 1 or 2 positive cells (pools 29 and 138) ( Figure 3).
  • One of these pools (138) was partitioned into smaller pools (20 pools of 1500 recombinants then 60 pools of 80 recombinants and finally 200 single clones), with autoradiographic screening at each stage, until a single cDNA clone was obtained which could transfer high capacity 125 I-hGM-CSF-binding to COS cells.
  • the individual cDNA clone in a second positive pool of DNA (pool 29) was ultimately identified as coding for the GM-CSF receptor by colony hybridization with the 1.8kbp insert of clone pGMR138. Plasmids encoding the cDNA inserts of clones 29 and 138 were designated pGMR29 and pGMR138 respectively.
  • the binding characteristics of the cloned GM-CSF receptor (clone 138; Example 3) when transfected to COS cells are shown in Fig 4.
  • the saturation binding isotherm for 125 I-hGM-CSF binding to transfected COS cells at 20°C showed a single class of binding site with an equilibrium dissociation constant of 6.8nM and 600,000 receptors per cell.
  • untransfected COS cells or COS cells transfected with vector alone showed no significant binding at these cell concentrations (3 to 7X10 4 cells/point).
  • binding of 125 I-hGM-CSF to transfected COS cells was determined in suspension (in binding medium with or without 20mM EDTA and 100 ⁇ g/ml chondroitin sulphate to prevent cell aggregation), or on adherent cells.
  • the apparent K D varied from 4 to 8nM, indicating that neither calcium nor adherence significantly altered the binding characteristics of the transfected receptor.
  • clone 29 is represented by nucleotides 1-1709 and clone 138 by nucleotides 7-1807.
  • the two sequences are otherwlse identical, except for a single silent base difference (G ⁇ A) in clone 29 at position 1148.
  • Each sequence encodes a large open reading (ORF) frame of 400 amino acids preceded by a short ORF of 22 amino acids.
  • the methionine codon beginning the larger reading frame is in a context which corresponds well to the consensus sequence (RCC ATG G) for translation initiation sites (Kozak, 1987), while the shorter ORF begins with a methionine codon in poor context.
  • the large ORF begins with a presumed signal peptide sequence of 22 amino acids, and residue Glu23 is assigned to the first amino acid of the mature protein by comparison with typical signal peptide cleavage sites (von Heijne, 1986).
  • the predicted 378 amino acid mature GM-CSF receptor is calculated to have a molecular weight of 43,728, which is approximately half the size of the receptor observed by cross linking of 125 I-hGM-CSF to HL60 cells and to COS-7 cells transfected with clone pGMR138 (Figure 5).
  • This difference between the predicted size of the core receptor polypeptide and the mature receptor on cells is probably due to the attachment of carbohydrate to the 11 potential N-linked glycosylation sites in the putative 297 amino acid extracellular domain, since we have shown that the mature receptor does not contain disulphide-linked subunits.
  • FIG. 7 A hydrophobicity plot ( Figure 7) suggests that a stretch of 27 uncharged amino acids extending from Gly320 to Phe346 ( Figure 6) represents a transmembrane domain. This putative transmembrane domain is followed by a 554 amino acid intracellular domain which begins with a short stretch of basic amino acids, a feature common to the cytosolic face, next to the membrane-spanning segments, of many transmembrane proteins. The 54 amino acid intracellular domain of the GM-CSF receptor shares no apparent homology with that of the IL6-R.
  • the predicted GM-CSF binding extracellular domain (amino acids 23-319) contains eleven cysteine residues, but these do not appear to form disulphide loops characteristic of receptors of the immunoglobulin superfamily (Sims et al, 1988; Yamasaki et al, 1988).
  • the short intracellular domain (54 amino acids) may serve a role in signal transduction. This domain has no apparent sequence homology with the catalytic domain of any growth factor receptor known to be a tyrosine kinase (Hanks et al, 1988). However, direct comparison of the GM-CSF receptor with that of the human IL-6 receptor (Yamasaki et al, 1988) revealed significant homology (Fig. 9).
  • the positions of four cysteine residues are approximately conserved (GM-CSF-R C 126 , C 136 , C 165 , C 178 vs. IL6-R C 121 , C 132 , C 165 , C 176 ), and these do not coincide with the two cysteine residues (C 47 and C 96 ) of the IL-6 receptor associated with the immunoglobulin-like domain (Yamasaki et al, 1988). Furthermore, there are patches of homology in the intracellular domain (Fig. 9) and a perhaps surprising similarity between the transmembrane domains of the two receptors (GM-CSF-R L 33 - L 342 vs.
  • IL6-R L 374 -L 386 including in each case the transmembrane cysteine residue.
  • the particular conservation of residues in the transmembrane regions may suggest a common ability to associate with other transmembrane proteins or lipids in their respective membranes. Indeed, a similarly placed cysteine residue in the transmembrane region of the Semliki Forest virus El spike protein, corresponding to the middle of the inner leaflet of the membrane, has been shown to be a site of palmitoylation (Schmidt et al, 1988).
  • the extracellular domain of the hGM-CSF receptor shows homology not only to that of the IL-6 receptor but also to those of the receptors for erythropoietin (D'Andrea et al, 1989), interleukin-2 (Hatekeyama et al, 1989), rat prolactin (Boutin et al, 1988) interleukin-4 (Mosley et al, 1989) and interleukin-3 (Itoh et al, 1990).
  • the regions of homology include the four cysteine residues mentioned above and a short amino acid sequence centred around the sequence Trp-Ser-X-Trp-Ser near the transmembrane region (Fig. 9).
  • a short ORF Prior to the long ORF encoding the GM-CSF receptor is a short ORF. This potentially encodes a polypeptide, although since its initiation methionine is in a context unrelated to the consensus sequence for good translation initiation (see above) this reading frame may not be translated (Kozak, 1986).
  • Such short ORFs 5' of the main receptor coding region are found in the hIL6 receptor cDNA (Yamasaki et al, 1988), and one of these (25 nucleotides upstream) in fact begins with a methionine codon in a context that is stronger than that which was assigned to begin the hIL6 receptor precursor. This suggests that these short ORFs might act to depress the translation of the main receptor coding regions.
  • cDNA clone pGMR138 was isolated from a human placental cDNA library, we tested whether or not mRNA corresponding to this transcript was also present in haemopoietic cells known to express the GM-CSF receptor.
  • HL-60 cells which are known to express high-affinity GM-CSF receptors, contain a 2.lkb transcript hybridizing at high stringency to the pGRM138 probe (tracks 3-5), whereas CEM T-lymphoblastoid cells and HepG2 hepatocellular carcinoma cells contain no detectable transcript hybridizing with this probe (tracks 1 and 2).
  • RNA from various haemopoietic and non-haemopoietic cells was undertaken, using PCR-based amplification of cDNA corresponding to various RNAs.
  • Such analyses e.g. Fig 8B
  • HeLa cells also display GM-CSF receptors, and have transcripts corresponding to this cDNA clone ( Figure 8B).
  • the cloned low-affinity receptor for human GM-CSF from placental cells described above, is introduced into a murine GM-CSF-dependent haemopoietic cell line (FDC-P1) using a retroviral vector, it retains its low-affinity phenotype but can nevertheless transmit the biological signals required for cell proliferation.
  • FDC-P1 murine GM-CSF-dependent haemopoietic cell line
  • the murine FDC-Pl haemopoietic cell line used does not proliferate in cultures containing 10 6 Units/ml of hGM-CSF, and no cells survive in such cultures.
  • hGM-R-FD lines Cloned cell lines
  • the parental FDC-P1 cell line usually exhibits a cloning efficiency in agar-medium of 60-100% when stimulated by mGM-CSF, forming large, tight, colonies.
  • hGM-R-FD lines maintained in hGM-CSF usually exhibited lower clonogenic potential (42 ⁇ 17%), and total colony numbers were similar in cultures stimulated by h or mGM-CSF (Figure 11).
  • the colonies characteristically had an irregular shape or were wholly dispersed, and maximal colony size was relatively small. Colony size was typically 2-4 times larger in parallel cultures stimulated by mGM-CSF. (For nine cell lines, mean colony size with mGM-CSF was 530 ⁇ 340 cells, versus 240 ⁇ 110 with hGM-CSF).
  • the dose-response curves of hGM-R-FD lines responding to stimulation by h or mGM-CSF indicated that responsiveness to hGM-CSF was 500-1000-fold lower than to mGM-CSF ( Figure 11, Table 1).
  • Cell lines grown using hGM-CSF were twofold more responsive to hGM-CSF than cell lines maintained in a mixture of m+hGM-CSF, although both types exhibited a similar responsiveness to mGM-CSF ( Figure 11, Table 1).
  • hGM-R-FD lines were assessed for their capacity to specifically bind 125 I-hGM-CSF and, whilst all of them showed significant binding, there was considerable variation in the extent of this binding (Table 1).
  • Clones maintained continuously in hGM-CSF alone showed higher average levels of bindlng than clones maintained in a mixture of hGM-CSF and mGM-CSF (Table 1).
  • hGM-CSF The binding of hGM-CSF at concentrations up to 1 ⁇ g/ml to the transfected receptor in hGM-R-FD clones for 30 min at 37°C did not affect the subsequent binding of 125 I-mGM-CSF to the native mGM-CSF receptor co-expressed on these cells.
  • mGM-CSF or mMulti-CSF at concentrations up to 0.5 ⁇ g/ml to their native receptors on hGM-R-FD cells at 37°C did not affect the subsequent binding of 125 I-hGM-CSF to the transfected hGM-CSF receptor.
  • the quantitative responsiveness of hGM-R-FD cells to hGM-CSF was 500-1000-fold lower than to mGM-CSF, but determination of the binding constants suggests that occupied hGM-CSF or mGM-CSF receptors might be equally efficient in transducing proliferative signals in murine FDC-Pl cells.
  • RNA from six sublines maintained with mGM-CSF contained no detectable hGM-R viral transcripts ( Figure 10B, tracks 2-7), compared with abundant hGM-R viral transcripts evident in sublines maintained with hGM-CSF (tracks 8-11 and other data not shown), suggesting that the alteration in such cells was at the transcriptional or immediately post-transcriptional level.
  • hGM-R-FD cell lines differed, depending on whether they were maintained in hGM-CSF alone or a mixture of hGM-CSF and mGM-CSF.
  • the former cell lines maintained a stable phenotype with equal clonogenicity in hGM-CSF or mGM-CSF, but clonogenicity was significantly lower than for cell lines maintained in mGM-CSF.
  • the cell lines maintained in a mixture of mGM-CSF and a low concentration of hGM-CSF showed a progressive loss of cells able to respond to stimulation by hGM-CSF.
  • the behaviour of cell lines maintained in hGM-CSF was not influenced by the site of viral integration.
  • the selection pressure exerted by culture with hGM-CSF alone maintained a constant level of expression of both viral genes (hGM-CSF-R and neoR), receptor expression and quantitative responsiveness to hGM-CSF.
  • the low clonogenicity and colony size exhibited by these cells suggested the continuous generation within lines maintained in hGM-CSF of progeny cells that had lost responsiveness to hGM-CSF. In the absence of any other stimulus, these cells irreversibly lost proliferative potential and could not be rescued by subsequent culture in mGM-CSF.
  • GM-CSF receptors of this invention A wide variety of potential therapeutic, diagnostic and preparative applications of GM-CSF receptors of this invention is envisaged, including, but not limited to, the following:

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Claims (35)

  1. Gereinigte Nucleinsäure, ausgewählt aus Einzelstrang-DNA, Doppelstrang-DNA, cDNA und RNA, die jeweils eine Nucleotidsequenz umfassen, die eine Nucleotidsequenz codiert oder dazu komplementär ist, die menschliche GM-CSF-Rezeptor oder ein Derivat davon mit der Fähigkeit, menschlichen GM-CSG zu binden, codiert, wobei der GM-CSF-Rezeptor definiert ist als ein glykosyliertes oder unglykosuliertes proteinartiges Molekül, umfassend in seiner Gesamtheit eine extrazelluläre Domäne, eine Transmembrandomäne und einen intracytoplasmatischen Rest, und die Fähigkeit hat, radioaktiv markierten GM-CSF oder Derivate davon spezifisch zu binden, wobei um diese Bindung unmarkierter GM-CSF konkurriert.
  2. Nucleinsäure nach Anspruch 1, wobei das Derivat davon mindestens 75 % Aminosäurehomologie mit dem natürlich auftretenden GM-CSF-Rezeptor aufweist.
  3. Nucleinsäure nach Anspruch 1 oder 2, wobei die Nucleinsäure eine DNA ist, die eine Nucleotidsequenz umfaßt, wie sie dargestellt ist in Figur 6B oder welche mindestens 75 % Homologie mit der Sequenz aus Figur 6B aufweist.
  4. Nucleinsäure nach einem der Ansprüche 1 bis 3, die zusätzlich eine weitere Nucleinsäuresequenz umfaßt, die benachbart ist zu der Sequenz, die den menschlichen GM-CSF-Rezeptor codiert.
  5. Nucleinsäure nach einem der Ansprüche 1 bis 4, wobei der GM-CSF-Rezeptor eine extrazelluläre Domäne umfaßt, die die Fähigkeit besitzt, GM-CSF zu binden.
  6. Rekombinantes DNA-Molekül, umfassend ein Gen, das den menschlichen GM-CSF-Rezeptor codiert, welcher die Aminosäuresequenz hat, wie sie in Figur 6B dargestellt ist, oder eine dazu homologe Sequenz, die ein Polypeptid codiert, das mindestens 75 % Sequenzidentität mit dem Rezeptor besitzt.
  7. Rekombinantes DNA-Molekül nach Anspruch 6, wobei das Polypeptid eine extrazelluläre Domäne, eine Transmembrandomäne und eine intrazelluläre Domäne umfaßt.
  8. Plasmid oder replizierbarer Vektor, umfassend eine Nucleinsäure gemäß der Definition in einem der Ansprüche 1 bis 5 oder rekombinantes DNA-Molekül nach Anspruch 6 oder 7.
  9. Rekombinante Zelle, umfassend eine Nucleinsäure gemäß der Definition in einem der Ansprüche 1 bis 5, oder ein rekombinantes DNA-Molekül nach Anspruch 6 oder 7.
  10. Rekombinante Zelle nach Anspruch 9, welche eine Affen-COS-Zelle ist.
  11. Rekombinante Zelle nach Anspruch 9, welche eine haematopoetische Zelle ist, wobei die Nucleinsäure einen Rezeptor für einen heterologen GM-CSF codiert.
  12. Rekombinante Zelle nach Anspruch 11, welche eine GM-CSF-abhängige haematopoetische Zelle ist.
  13. Rekombinanter oder synthetischer menschlicher GM-CSF-Rezeptor oder ein Derivat davon mit der Fähigkeit, menschlichen GM-CSF zu binden, im wesentlichen frein von anderen Proteinen, wobei der GM-CSF-Rezeptor definiert ist als ein glykosyliertes oder unglykosyliertes proteinartiges Molekül, umfassend in seiner Gesamtheit eine extrazelluläre Domäne eine Transmembrandomäne und einen intracytoplasmatischen Rest, und die Fähigkeit hat, radioaktiv markierten GM-CSF oder Derivate davon spezifisch zu binden, wobei um diese Bindung unmarkierter GM-CSF konkurriert.
  14. Rekombinanter oder synthetischer menschlicher GM-CSF-Rezeptor nach Anspruch 13, wobei das Derivat mindestens 75 % Aminosäurehomologie mit dem natürlich vorkommenden GM-CSF-Rezeptor aufweist.
  15. Rekombinanter oder synthetischer GM-CSF-Rezeptor nach Anspruch 13, umfassend eine der Aminosäuresequenzen, wie die in Figur 6B dargestellt ist, oder eine, die zu den in Figur 6B dargestellten Aminosäuren eine mindestens 75 %ige Homologie aufweist.
  16. Rekombinanter oder synthetischer GM-CSF-Rezeptor nach einem der Ansprüche 13 bis 15, der ein Fusionsprotein oder ein Hybridprotein ist.
  17. Rekombinanter oder synthetischer GM-CSF-Rezeptor nach einem der Ansprüche 13 bis 16, wobei der GM-CSF-Rezeptor die extrazelluläre Domäne umfaßt, die die Fähigkeit hat, GM-CSF zu binden.
  18. GM-CSF-Rezeptor, der durch eine rekombinante Zelle gemäß der Definition in einem der Anspüche 9 bis 12 produziert wird.
  19. Antikörper oder Antigen-bindendes Fragment davon, wobei der Antikörper oder das Fragment einen rekombinanten oder synthethischen GM-CSF-Rezeptor nach einem der Ansprüche 13 bis 18 spezifisch bindet.
  20. Arzneimittel, umfassend eine rekombinanten oder synthetischen GM-CSF-Rezeptor nach einem der Ansprüche 13 bis 18 oder einen Antikörper oder ein Antigen-bindendes Fragment davon nach Anspruch 19.
  21. Verwendung eines rekombinanten oder synthethischen GM-CSF-Rezeptors oder eines Derivates davon gemäß der Definition in einem der Ansprüche 13 bis 18 oder eines Antikörpers oder Antigen-bindenden Fragmentes davon gemäß der Definition in Anspruch 19 für die Herstellung eines Medikamentes zur Verwendung in der Behandlung von mit GM-CSF in Zusammenhang stehenden Krakheiten.
  22. Verwendung eines GM-CSF-Rezeptors oder eines Derivates oder eines Antikörpers oder Antigen-bindenden Fragments davon gemäß der Definition in Anspruch 19 für die Herstellung eines Mittels zur Verwendung bei der Modulation der Proliferation, Differenzierung oder der funktionellen Aktivierung von GM-CSF-Stimulations-sensitiven Zellen in einem Säuger.
  23. Verfahren zur Beschaffung von Information zur Verwendung in der Diagnose einer mit GM-CSF in Zusammenhang stehenden Krankheit in einem Säuger, umfassend das Inkontaktbringen eines Gewebes oder einer Flüssigkeit dieses Säugers in vitro mit einer Nucleinsäure oder einem rekombinanten DNA-Molekül gemäß der Definition in einen der Ansprüche 1 bis 7, einer rekombinanten Zelle gemäß der Definition in einem der Ansprüche 9 bis 12, einem rekombinanten oder synthetischen GM-CSF-Rezeptor gemäß der Definition in einem der Ansprüche 13 bis 18 oder einem Antikörper oder Antigen-bindenden Fragment davon gemäß der Definition in Anspruch 19, und den Nachweis des dabei erzeugten Produkts.
  24. Verwendung einer Nucleinsäure oder eines rekombinanten DNA-Molekül gemäß der Definition in einem der Ansprüche 1 bis 7, einer rekombinanten Zelle gemäß der Definition in einem der Ansprüche 9 bis 12, eines rekombinanten oder synthetischen GM-CSF-Rezeptors gemäß der Definition in einem der Ansprüche 13 bis 18, oder eines Antikörpers oder Antigen-bindenden Fragments davon gemäß der Definition in Anspruch 19 für die Herstellung eines Mittels zur Verwendung in der Diagnose einer mit GM-CSF in Zusammenhang stehenden Krankheit in einem Säuger.
  25. Verfahren nach Anspruch 23 oder Verwendung nach Anspruch 24, wobei die mit GM-CSF in Zusammenhang stehende Krankheit Krebs, ein Tumor oder Leukämie ist, welche/welcher durch GM-CSF stimulierbare Zellen verursacht wird oder damit assoziiert ist.
  26. Verfahren zum Screenen einer cDNA-Bank nach einem cDNA-Fragment, das den GM-CSF-Rezeptor codiert, wobei der GM-CSF-Rezeptor definiert ist als ein glykosyliertes oder unglykosyliertes proteinartiges Molekül, umfassend in seiner Gesamheit eine extrazelluläre Domäne, eine Transmembrandomäne und einen intracytoplasmatischen Rest, und die Fähigkeit hat, radioaktiv markierten GM-CSF oder Derivate davon spezifisch zu binden, wobei um die Bindung unmarkierter GM-CSF konkurriert, umfassend folgende Schritte:
    (a) Konstruktion einer cDNA-Bank,
    (b) Herstellung von cDNA-Fragmenten davon,
    (c) Transfektion dieser Fragmente in Säugerwirtszellen,
    (d) Inkubation der transfizierten Zellen mit markiertem GM-CSF,
    (e) Identifizierung von Populationen transfizierter Zellen, die den markierten GM-CSF binden,
    (f) Hersetellung von Wirtszellclonen, die mit solchen cDNA-Fragmenten transformiert sind, die die Fähigkeit besistzen, Säugerwirtszellen zur Bindung von markiertem GM-CSF zu veranlassen, und die Isolierung dieser Clone.
  27. Verfahren zur Herstellung des GM-CSF-Rezeptors, umfassend die Schritte des Züchtens einer rekombinanten Zelle nach einem der Ansprüche 9 bis 12 in einem geeigneten Nährmedium und Gewinnung des GM-CSF-Rezeptors.
  28. Verfahrens zur Herstellung einer DNA-Sequenz, die den GM-CSF-Rezeptor codiert, wobei der GM-CSF-Rezeptor definiert ist als glykosyliertes oder unglykosyliertes proteinartiges Molekül, umfassend in seiner Gesamtheit eine extrazelluläre Domäne, eine Transmembrandomäne und einen intracytoplasmatischen Rest, und die Fähigkeit hat, radioaktiv markierten GM-CSF oder Derivate davon spezifisch zu binden, wobei um die Bindung unmarkierter GM-CSF konkurriert, umfassend die folgenden Schritte:
    (a) Bereitstellung einer cDNA-Bank aus einer Quelle, die den GM-CSF-Rezeptors exprimiert;
    (b) Herstellung von cDNA-Fragmenten davon;
    (c) Transfektion der Fragmente in Wirtszellen;
    (d) Identifizierung von Wirtszellen, die den GM-CSF-Rezeptor exprimieren;
    (e) Gewinnung der Wirtszellen, die den GM-CSF-Rezeptor exprimieren; und
    (f) Gewinnung der DNA aus den in Schritt (e) erhaltenen Wirtszellen.
  29. Verfahren nach Anspruch 28, wobei Schritt (d) nach dem Verfahren von Anspruch 26 ausgeführt wird.
  30. Verfahren nach einem der Ansprüche 26, 28 oder 29, wobei die cDNA-Bank aus menschlicher Plazenta stammt.
  31. Plasmide pGMR138 und pGMR29, gemäß der Definition in Beispiel 3.
  32. Zellinie hGM-R-FD gemäß der Definition in Beispeil 8.
  33. Verfahren zum Screenen von Verbindungen auf die Fähigkeit, die Bindung von GM-CSF zu verstärken oder zu reduzierten, umfassend die Schritte des Inkontaktbringens einer Zellinie, die rekombinante GM-CSF-Rezeptoren trägt, mit GM-CSF, der mit einem nachweisbaren Marker markiert ist, in Gegenwart eines zu testenden Stoffes, und der Messung, ob die Bindung von GM-CSF an die transfizierten Zellen im Vergleich zur Bindung einer Bezugsverbindung verstärkt oder reduziert ist, wobei der GM-CSF-Rezeptor definiert ist als ein glykosyliertes oder unglykosyliertes proteinartiges Molekül, umfassend in seiner Gesamtheit eine extrazelluläre Domäne, eine Transmembrandomäne und einen intracytoplasmatischen Rest, und die Fähigkeit hat, radioaktiv markierten GM-CSF oder Derivate davon spezifisch zu binden, wobei um die Bindung unmarkierter GM-CSF konkurriert.
  34. Verfahren nach Anspruch 33, wobei die verwendete Zellinie hGM-R-FD gemäß der Definition in Beispiel 8 ist.
  35. Nucleinsäuresonde, deren Sequenz in der in Figur 6B dargestellten Sequenz enthalten oder dazu komplementär ist.
EP90912329A 1989-08-11 1990-08-10 Rezeptor für granulozyten-macrophagen-koloniestimulierungsfaktor und seine derivate Expired - Lifetime EP0486572B1 (de)

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AU5743/89 1989-08-11
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AUPK001490 1990-05-08
AU14/90 1990-05-08
PCT/AU1990/000342 WO1991002063A1 (en) 1989-08-11 1990-08-10 Improvements in granulocyte-macrophage colony-stimulating factor receptor and derivatives thereof

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WO1994009149A1 (en) * 1990-08-10 1994-04-28 Amrad Corporation Limited Monoclonal antibody
AU673858B2 (en) * 1992-10-09 1996-11-28 Amrad Operations Pty. Limited Monoclonal antibody
AU5611394A (en) * 1992-11-19 1994-06-08 Dana-Farber Cancer Institute Antibodies for gm-csf receptor and uses thereof
CA2265915C (en) * 1996-09-10 2012-11-20 Theodor-Kocher Institute Cxcr3 chemokine receptor, antibodies, nucleic acids, and methods of use
US6140064A (en) 1996-09-10 2000-10-31 Theodor-Kocher Institute Method of detecting or identifying ligands, inhibitors or promoters of CXC chemokine receptor 3
AU2074101A (en) * 1999-12-08 2001-06-18 Millennium Pharmaceuticals, Inc. Compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer
US7247618B2 (en) * 2001-04-30 2007-07-24 Tripathi Rajavashisth Methods for inhibiting macrophage colony stimulating factor and c-FMS-dependent cell signaling
WO2005038055A1 (en) * 2003-09-22 2005-04-28 Nathaniel Tue Tran Multiplexing array techniques
EP2423230B1 (de) * 2006-03-27 2013-05-08 Medimmune Limited Bindeelement für den GM-CSF-Rezeptor
US8475796B2 (en) 2008-12-22 2013-07-02 University Of Melbourne Method of treating pain using an antagonist of GM-CSF
ES2572368T5 (es) 2008-12-22 2021-12-16 Univ Melbourne Tratamiento de la artrosis
CA2775155A1 (en) * 2009-10-01 2011-04-07 Csl Limited Method of treatment of philadelphia chromosome positive leukemia
KR20140103122A (ko) 2011-11-17 2014-08-25 넨키 인스티튜트 오브 익스페리멘탈 바이올로지 신경교종을 치료하기 위한 조성물 및 방법
DK2932264T3 (da) * 2012-12-14 2019-05-06 Biontech Rna Pharmaceuticals Gmbh Nye MHC-uafhængige tumor-associerede antigener
WO2016090369A1 (en) 2014-12-05 2016-06-09 City Of Hope Cs1 targeted chimeric antigen receptor-modified t cells

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AU594014B2 (en) * 1984-03-21 1990-03-01 Research Corporation Technologies, Inc. Recombinant DNA molecules
US5109119A (en) * 1989-06-06 1992-04-28 Schering Corporation Crystalline r-h-gm-csf and method
US5112961A (en) * 1990-07-18 1992-05-12 Schering Corporation Dna encoding subunits of a high affinity gm-csf receptor

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US6136957A (en) 2000-10-24
DK0486572T3 (da) 1998-05-04
DE69031914T2 (de) 1998-07-30
EP0486572A1 (de) 1992-05-27
AU637133B2 (en) 1993-05-20
CA2064814C (en) 2004-06-08
CA2064814A1 (en) 1991-02-12
SG59954A1 (en) 1999-02-22
JP3267966B2 (ja) 2002-03-25
JPH04507344A (ja) 1992-12-24
US5726036A (en) 1998-03-10
AU6189690A (en) 1991-03-11
WO1991002063A1 (en) 1991-02-21
ATE161883T1 (de) 1998-01-15
DE69031914D1 (de) 1998-02-12

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